In recent studies it has been revealed that, immune competent cells with macrophage activity such as the Kupffer cells have a remarkably sensitive and potent osmoregulation, see e.g. Biochem J. 1995, Vol. 312, pag. 135-142, F Zhang et al. The studies suggest that cell volume homeostasis is a critical factor for the cellular function of Kupffer cells. This type of organic osmolytes need to be non-perturbing solutes that do not interfere with protein function even when occurring at high intracellular concentrations. Such a prerequisite may explain why only a few classes of organic compounds, viz. polyols (e.g. inositol and sorbitol), methylamines (betaine, .alpha.-glycerophosphorylcholine) and certain amino acids such as taurine have evolved as osmolytes in living cells. In mammals, osmolytes have been identified in astrocytes, renal medulla cells and lens epithelia. The need for osmolytes in renal medulla cells is obvious, because ambient medullary osmolarity can increase up to 3800 mosmol/l during antidiuresis and decrease to 170 mosmol/l during diuresis. In the antidiuretic state (high extracellular osmolarity), intracellular osmolarity increases in renal medullary cells as the result of the intracellular accumulation of inositol and betaine which are taken up via sodium ion dependent transporters. These sodium ion dependent transporters are induced upon hyperosmotic exposure in renal cells and astrocytes. Recent studies with Madine-Darby canine kidney (MDCK) cells have identified a hypertonic stress-responsive element in the 5'-flanking region of the mammalian BGT-1 gene (betaine transporter).
In a study disclosed in FEBS Letters, 1995, Vol. 377, pages 47-50, U Warskulat et al., betaine is identified as osmolyte in mouse macrophages. The betaine uptake in mouse macrophages was significantly stimulated when the cells were exposed to a hyperosmotic (405 mosm/l) medium. From the results of this study it was concluded that betaine availability could be a potential site for the regulation of macrophage cell function.
Certain organic osmolytes have previously been suggested in the International Patent Application WO 91/14435 as supplements to protect cells in a dehydrated environment from volume changes. Also in Biocbem. Journal, 1992, Vol. 282, pages 69-73, it is demonstrated that SV-3T3 cells (fibroblasts) subjected to hyperosmotic conditions may retain normal function in terms of rate of cell proliferation and protein synthesis in the presence of an osmolyte. Even if these publications may consider a therapeutic utility of certain osmolytes, there are no disclosures of how osmolytes can affect cells which mediates pathological events resulting from ischemia, hypoxia or oxidative stress, both during hyperosmolar conditions and in conditions with normal osmolarity.
Organ transplantation has become an established therapy for end stage liver and heart disease, although primary graft non-function or dysfunction is serious clinical problem. Cold ischemic storage and the following reperfusion of the donated organ are identified as major contributors to failing primary graft function and is shown to have a detrimental impact on endothelial and immune competent cells, injuries to the endothelial cells precipitates a malfunction vascular system and consequently, an inadequate oxygen and substrate delivery, as well as an impaired waste product clearance. Furthermore, the challenged endothelium enhances the expression of adhesive molecules facilitating the binding and infiltration of immune competent cells in the tissue area at risk. Immune competent cells respond to ischemia and reperfusion by producing a number of biologically toxic mediators, again leading to the dysfunction of surrounding cells, including the vascular endothelium and in certain cases the whole organ. The early organ dysfunction is considered to originate from injuries of endothelial cells resulting in inadequate oxygen and substrate delivery as well as reduced waste product clearance. Beyond transplantation injuries, resulting from ischemia and reperfusion, these are a well recognized clinical problems in, for example, myocardial infarction and the following thrombolytic treatment. As disclosed in Laboratory Investigation, 1996, Vol. 74, No. 1, p. 86 (J Kajstura et al.), both myocardial ischemia and hypoxia can induce cell death, such as programmed cell death (apoptosis) in the heart following myocardial infarction which may lead to massive loss of cells and further organ damages.
In the liver, the inflammatory response to ischemia and reperfusion is suggested to be primarily mediated by resident macrophages, the Kupffer cells, while the heart in such a situation suffers from invading immune competent cells which might cause persistent injuries.
It would consequently be highly desirable to find a suitable therapy to preserve or improve the endothelial cell function and diminish the inflammatory response of the immune competent cells during and after the mentioned complications, as well as form a protection against cell death.
In response to ischemia/reperfusion and inflammatory mediators, endothelial and immune competent cells produce oxygen free radicals which exert a detrimental metabolic load on exposed cells termed oxidative stress. The oxidative stress precipitates severe damages to biological molecules, especially to DNA, lipids and proteins. The protection against oxidative stress and hence the salvage of tissues and organs might be achieved only partially by supplying antioxidants and ensuring an adequate level of antioxidant enzymes. It would therefore also be desirable to be able to provide a therapy which also is useful for improving the protection of cells against damages originating from oxidative stress.
In the International patent application WO 92/15546 certain osmolytes, such as taurine, which are capable of crossing the blood brain barrier, are suggested in the protection of cells being at risk to be damaged from lactic acidosis from oxygen deficiency. In this publication, however, the osmolyte exert its beneficial effect by providing buffering action and not by directly acting on specific cells in order to modulate their response to the disorderly event. Furthermore, the osmolyte taurine has been suggested to have certain beneficial effects to heart in Japanese Circ. Journ. 1992, Vol. 56, p. 95 (J Azuma et al.) following congestive heart failure. It is concluded that taurine possibly contributes to a regulation of the myocardial calcium uptake and thus may increase the myocardial activity. According to the present invention, it has been surprisingly found that certain osmolytes, such as betaine and taurine, have a powerful capacity to maintain the cellular integrity in specific cells, and thereby the organ function, subjected to a depletion of oxygen in an anoxia model or oxidative stress, as demonstrated in an isolated, perfused liver. The present invention shows that selected osmolytes can be employed as important regulators of endothelial and immune competent cell function. The osmolytes have a capacity to protect these cell types or to affect such cells to modulate their response to the mentioned complications and thereby maintaining the function of vital organs challenged by pathologic events, such as an inadequate blood supply.
The failing liver is an early event in sepsis and accompanied by raised enzyme leakage from the liver, for example lactate dehydrogenase (LDH) which indicates a compromised cellular integrity. As a sign of an adequate treatment, the hepatic function and enzyme leakage is restored to near normal levels within days. This course of pathological events and the impact of a successful treatment, reflects the clinical importance of the marked decrease in LDH leakage in response to osmolyte treatment following anoxia, as will be described in the present invention.
Consequently, it is an object of the present invention to preserve and improve the endothelial cell function and diminish the inflammatory response of the immune competent cells by a supplementation of an effective amount of certain osmolytic agents. It is also an object of the present invention to, by means of an osmolyte therapy, to improve the capacity of the tissue to resist oxidative stress, in order to prevent and treat damages resulting from such a condition and thereby improve the possibility of organ protection and rescue.
The present invention demonstrates that otherwise metabolically inert osmolytes have a high potency in protecting organs or tissues from such damages and dysfunctions resulting from ischemia and reperfusion, hypoxia or oxidative stress.